Introduction

Diabetic cardiomyopathy is one of the leading causes of increased morbidity and mortality in the diabetic population. It is attributed to changes in the composition of the extracellular matrix (ECM) with enhanced cardiac fibrosis, increased cardiac cytokine levels (Ma et al. 2012; Tschope et al. 2005), and myocyte hypertrophy (Frustaci et al. 2000).

In humans (Jarrett 1989) and in the streptozotocin (STZ) rat model of type 1 diabetes (Mihm et al. 2001), cardiomyopathy is associated with an initial diastolic dysfunction followed by altered contractile performance. Cardiac cytokine such as transforming growth factor (TGF)-β, is a potent profibrotic marker involved in the development of cardiac fibrosis and cardiac failure and regulating the synthesis of the matrix-associated protein fibronectin (Feldman et al. 2000; Sun et al. 2004; Torre-Amione et al. 1996). Furthermore, a dysregulation of collagen-degrading matrix metalloproteinases (MMPs) and their tissue inhibitors is supposed to be a hallmark for myocardial fibrosis in diabetes. However, there is little information regarding MMPs and their tissue inhibitors in diabetic cardiomyopathy.

Reactive oxygen species (ROS), particularly superoxide anions (O2 −), have emerged as key mediators in cardiac pathophysiology, involved in the development of hypertrophy, fibrosis, and contractile dysfunction (Date et al. 2002; Dhalla et al. 1996). Increased ROS have been reported to be involved in oxidative myocardial injury and exacerbates diabetic cardiomyopathy (Ma et al. 2006; Yu et al. 2012) and nephropathy (Luo et al. 2010). In systemic hypertension, the level of O2 − production parallel changes in the vascular wall (Dobrian et al. 2001).

Numerous experimental studies demonstrated that an antioxidant intervention at the beginning of reperfusion reduces all major detrimental manifestations of myocardial infarction (Li and Jackson 2002).

4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) is a stable piperidine nitroxide of low molecular weight and cell membrane-permeable superoxide dismutase (SOD) mimetic, was shown to reduce systemic blood pressure, vascular resistance, and medial hypertrophy of systemic resistance arteries in different rat models of systemic hypertension (Schnackenberg et al. 1998). Tempol blocked the cardiac fibrosis, myofibroblast proliferation, and cardiac collagen accumulation produced by an infusion of angiotensin II (Zhao et al. 2008). However, a paucity of information is only available regarding the role of tempol and its specific mechanism(s) involved in management of diabetic cardiomyopathy.

Therefore, the present study was designed to examine whether tempol can ameliorate the cardiac fibrosis associated with diabetes in rat model and to address the precise mechanism(s) underlying this effect.

Materials and methods

Animals

Adult male Sprague–Dawley rats (220–250 g, 10- to 12-week old) were housed at room temperature with 12:12-h light/dark cycles and were given food and water ad libitum.

Experiments were conducted in accordance with the international ethical guidelines for animal care of the US Naval Medical Research Centre, Unit No. 3, Abbaseya, Cairo, Egypt, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The adopted guidelines are in accordance with "Principles of Laboratory Animals Care" (NIH publication no. 85-23, revised 1985). The study protocol was approved by members of "The Research Ethics Committee" and by the Pharmacology and Toxicology Department, Faculty of Pharmacy, Minia University, Egypt.

Induction of diabetes

STZ (Sigma Aldrich, St Louis, MO, USA), freshly dissolved in 10 mmol/L citrate buffer, pH 4.5), was intraperitoneally injected at a single dose of 50 mg/kg for diabetes induction (Coskun et al. 2005). Rats with blood glucose levels of ≥300 mg/dL were considered to be diabetic.

Experimental design

Rats were randomly divided into four groups of seven rats each, as follows:

  1. 1.

    Control group: normal nondiabetic rats, received the same volume of the solvent.

  2. 2.

    Control + tempol-treated group: rats treated with tempol (Sigma Aldrich, St Louis, MO) in a dose of 18 mg kg−1 day−1, dissolved in saline (Castro et al. 2009) by gavage for 8 weeks.

  3. 3.

    Diabetic group (STZ group): rats were treated with STZ as described above.

  4. 4.

    Diabetic group treated with the tempol (STZ + tempol): rats were orally treated with tempol at a dose of 18 mg/kg for 8 weeks as described above with control group. The animals were maintained in their respective groups for 8 weeks.

Hemodynamic and cardiac parameters

Rats were anesthetized by intraperitoneal administration of pentobarbital at 50 mg/kg body weight; systolic blood pressure (SBP) and heart rate were measured by the tail cuff method (Pressure Meter, Model LE 5001; Panlab, Barcelona, Spain).

After completion of the hemodynamic measurements, serum glucose, lactate dehydrogenase (LDH), and creatine kinase (CK-MB) were spectrophotometrically estimated (Shimadzu, Kyoto, Japan).

Subsequent to blood collection, the hearts were immediately removed and placed in ice-cold Krebs solution. Hearts were weighed and the heart weight/body weight (HW/BW) ratio was calculated. Then ventricles (left and right ventricles) were isolated. A portion of the left ventricle apex was fixed in 10 % neutral-buffered formalin and processed for histopathological and immunohistochemical analysis. The remaining ventricular tissues were homogenized in cold potassium phosphate buffer (0.05 mM, pH 7.4) and centrifuged at 5,000×g for 10 min at 4 °C. The supernatant was kept at −80 °C for subsequent measurements. Total protein concentration was also determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Chemicals).

Western blot analysis

Protein expression of collagen types IV and fibronectin were determined in cardiac tissues using Western blot analysis. Frozen left ventricular tissues were homogenized in ice-cold lysis buffer containing: 20 mmol/L Tris·HCl, 140 mmol/L NaCl, 1 mmol/l EDTA, complete miniprotease inhibitor cocktail, 1 % Triton X-100, 0.1 % sodium dodecyl sulfate (SDS), 1 % sodium deoxycholate, 1 mmol/l NaF, and 1 mmol/L orthovanadate, and pH 7.8. Following protein concentration estimation using BCA, equal amounts of membrane protein (20 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After incubation with blocking solution (4 % nonfat milk, Sigma), membranes were incubated with 1:1,000 collagen IV, fibronectin (Abcam, Cambridge, UK) antibodies for 2 h at room temperature. Membranes were washed and then incubated with a 1:3,000 dilution of second antibody (Amersham Life Science) for 1 h, and the membranes were detected with the enhanced chemiluminescence system (Amersham Life Science). To correct differences in protein loading, the membranes were washed and reprobed with 1:1,000 dilution monoclonal antibodies to human ß-actin (Abcam, Cambridge, UK). Relative intensities of protein bands were analyzed by scanner and quantified by AIDA Image Analyzer software.

Determination of MMP-2 activity

Cardiac MMP levels, including MMP-2 were determined in the heart using gelatin zymography as described previously (Hayashidani et al. 2003). Frozen left ventricular tissue was homogenized in 1 mL of an ice-cold extraction buffer containing cacodylic acid (10 mmol/L), NaCl (0.15 mmol/L), ZnCl2 (20 mmol/L), NaN2 (1.5 mmol/L), and 0.01 % Triton X-100 (pH 5.0). The homogenate was centrifuged (4°C, 10 min, 10,000×g), and the supernatant decanted and saved on ice. The pH levels of the samples were adjusted to pH 7.5 using Tris (1 mmol/L). The extracted samples were then aliquoted and stored at −80 °C until the time of assay. The cardiac extracts were then directly loaded onto electrophoretic gels (SDS-PAGE) containing 1 mg/mL of gelatin under nonreducing conditions. The cardiac extracts at a final protein content of 5 μg were loaded onto the gels using a 3:1 sample buffer (10 % SDS, 4 % sucrose, 0.25 mmol/L Tris-HCl, and 0.1 % bromophenol blue). The gels were run at 15 mA/gel through the stacking phase (4 %) and at 20 mA/gel for the separating phase (10 %), whereas the running buffer temperature was maintained at 4 °C. After SDS-PAGE, the gels were washed twice in 2.5 % triton X-100 for 30 min each, rinsed in water, and incubated for 24 h in a substrate buffer at 37 °C (50 mmol/L Tris-HCl, 5 mmol/L, CaCl2, and 0.02 % NaN3 (pH 7.5)). After incubation, the gels were stained with Coomassie brilliant blue. The gelatinolytic activity was detected as a clear band against a blue background (arbitrary unit per centimeter) and analyzed as relative optical densities using AIDA imaging software.

Estimation of cardiac TGF-β level

The fibrogenic cytokine, TGF-β content in left ventricular tissues was assessed by enzyme-linked immunosorbent assay by microplate ELISA reader (Spectra III Classic, Tecan, Salzburg, Austria) using rat TGF-β assay kit (MyBioSource, CA, USA) following the instructions of the manufacturer and based on previously described method (Date et al. 2002). Results are expressed as nanograms per milligram of protein.

Measurement of ROS production and SOD enzyme activity

Left ventricular tissues were homogenized in a cold Krebs–HEPES buffer (10 mmol/L glucose, 0.02 mmol/L Ca-Tritriplex, 25 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, 120 mmol/L NaCl, 1.6 mmol/L CaCl2·2H2O, 1.2 mmol/L MgSO4·7H2O, and 5 mmol/L KCl (pH 7.4)). ROS production was measured using lucigenin-derived chemiluminescence as previously described (Taye et al. 2010). ROS production was measured using lucigenin-enhanced chemiluminescence (5 μmol/L) to minimize the redox cycling, incubated for 20 min. The reaction was started by the addition of NADPH (100 μmol/L), and the relative light units of chemiluminescences were measured over a period of 30 min using a lumencense spectrometer (Perkin-Elmer Ltd.,UK). Results were expressed as counts per minute and normalized to the protein content in each sample. Conversely, the antioxidant enzyme activity of cardiac SOD was assessed spectrophotometrcally using SOD kit (Biodiagnositc Company, Egypt).

Histopathological examination

The left ventricle from the heart of each animal was dissected out then fixed in buffered formalin for 12 h and processed for histopathological examination. Four micrometer-thick paraffin sections were stained with hematoxylin and eosin for light microscopic examination. A minimum of six fields for each heart left ventricle section were examined and assigned for changes by an observer blinded to the treatments of the animals. A semiquantitative evaluation of cardiac lesions and inflammatory alteration were scored for each group according to the previously described modified histological scoring protocol (Kodavanti et al. 2008).

Immunolocalization of TGF-β

The left ventricular tissues were cut into 4-mm sections and then fixed in paraformaldehyde (4 %, w/v) for 30 min. After being washed in phosphate-buffered saline, the sections were incubated with the primary antibody (anti-TGF-β antibody, Santa Cruz, CA, USA) at the dilution of 1:500 for an overnight incubation at 4 °C. The sections were incubated with Poly HRP DAB Kit (Genemed Biotechnologies, CA, USA) to visualize any antigen–antibody reaction in the tissues. Immunohistochemical evaluation was carried out using image analysis software (Image J, 1.46a, NIH, USA).

Statistical analysis

Data are expressed as means ± SEM (standard error of the mean) and were analyzed using the unpaired Student's t test for comparisons between two groups and by one-way ANOVA followed by the Bonferroni' s test for multiple comparisons. A probability value (P) below 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 5.

Results

General characteristics and effects of tempol treatment on diabetic rats

Throughout the 8-week study period, body weight was significantly decreased in diabetic rats compared with the control group. Administration of tempol increased the body weight of the diabetic rats. However, heart weight indexed to body weight (HW/BW) was significantly increased in diabetic rats compared with the control group. Administration of tempol reduced the HW/BW ratio of the diabetic rats (Table 1).

Table 1 Effect of tempol on the basal and hemodynamic characteristics of control and STZ-diabetic rats
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Furthermore, glucose levels were significantly elevated in STZ-treated group, however, tempol treatment has no significant effect in this setting (Table 1).

Effect of tempol on some hymodynamic and cardiac parameters

The untreated diabetic rats exhibited elevated SBP and reduced heart rate as compared with the control rats. However, treatment with tempol normalized both blood pressure and heart rate in the diabetic group (Table 1). Tempol significantly improved the disrupted parameters (Table 1). Furthermore, STZ-induced diabetes revealed a significant increase in the serum levels of LDH and CK as compared with control rats. Treatment with tempol significantly reduced the elevated levels of those cardiac markers in diabetic rats (Table 1).

Tempol attenuated collagen IV and fibronectin protein levels

To assess the alterations of profibrotic mediators involved in interstitial fibrosis, myocardial levels of ECM, MMPs, and TGF-β were determined.

Western blot analysis revealed a significant (P < 0.05) upregulation in both collagen IV and fibronectin protein levels (profibrotic markers) in cardiac diabetic rats compared with the controls. Tempol administration could reverse these changes (Fig. 1a, b).

Fig. 1
figure 1

Effect of tempol (18 mg kg−1 day−1, orally) on collagen IV and fibronectin protein expression in the left ventricles of STZ-induced diabetic rats. a Representative Western blots showing effect of tempol on collagen IV in the left ventricle. Bar graphs represent the quantitative difference in expression of collagen IV. b Representative Western blots displaying effect of tempol on fibronectin in the left ventricle. Bar graphs represent quantitative difference in expression of collagen IV. Values for each bar are the mean ± SEM for seven rats per group normalized to β-actin as an internal control and expressed as percent of control; *P < 0.05 vs., control group; #P < 0.05 vs. control + tempol group; ƒP < 0.05 vs. STZ-treated group

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Tempol normalized MMP-2 gelatinolytic activity

The gelatinolytic activity of MMP-2 was significantly decreased in the STZ-treated group (P < 0.05) compared with the control group. Tempol treatment normalized the disrupted poteolytic activity in diabetic rats (Fig. 2).

Fig. 2
figure 2

Effect of tempol (18 mg kg−1 day−1, orally) on MMP-2 activity in the left ventricles of STZ-induced diabetic rats. MMP-2 activity showing decreased proteolitic activity in cardiac tissues of diabetic rats, almost normalized in diabetic rats treated with tempol (STZ-tempol) group compared with nondiabetic controls by zymography. Values for each bar are the mean ± SEM for seven rats, expressed as percent of control; *P < 0.05, STZ-treated group vs. control group; #P < 0.05 vs. control + tempol-treated group; ƒP < 0.05 vs. STZ-treated group

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Tempol decreased cardiac TGF-β level

TGF-β level was significantly (2-fold, P < 0.05) increased in cardiac tissues of STZ-treated rats compared with the control group. Tempol administration significantly reduced the elevated level of TGF-β almost to the normal level (Fig. 3).

Fig. 3
figure 3

Effect of tempol (18 mg kg−1 day−1, orally) on TGF-β content in left ventricles of STZ-induced diabetic rats. Cardiac TGF-β content was measured using enzyme-linked immunosorbent assay showing increased levels of TGF-β in diabetic rats and tempol treatment normalized these changes. Values for each bar are the mean ± SEM for seven rats, expressed as percent of control; *P<0.01 vs. control group; #P < 0.01 vs. control + tempol-treated group; ƒP < 0.05 vs. STZ-treated group

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Effect of tempol on oxidative stress biomarkers

ROS production was significantly increased in the homogenates of cardiac diabetic rats as compared with control while tempol treatment prevented these changes (Fig. 4a). The cardiac activity of SOD as a major endogenous antioxidant enzyme was significantly attenuated in cardiac tissues of diabetic rats (Fig. 4b). Tempol restored the cardiac SOD activity in the cardiac tissues of diabetic rats close to the normal level.

Fig. 4
figure 4

Effect of tempol (18 mg kg−1 day−1, orally) on ROS production and the SOD activity in left ventricles of STZ-induced diabetic rats. a ROS production, measured by lucigenin-enhanced chemiluminescence assay was significantly increased in diabetic rats as compared with control. b The cardiac SOD activity was significantly lower in the diabetic rats than in control rats and tempol treatment inhibited these changes. Values for each bar are the mean ± SEM for 7 rats per group, expressed as percent of control; *P < 0.01 vs. control group; #P<0.05 vs. control + tempol-treated group; ƒP < 0.05 vs. STZ-treated group

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Histopathological and immunohistochemistry examination

Photomicrographic analysis revealed effects of tempol on histopathological alterations induced by STZ in the left ventricular tissues (Table 2). Picture of heart in control rats showed normal histological structure of the cardiac tissues (Fig 5a, b). Heart of diabetic rat exhibits vaculations of cardiac myocytes associated with inflammatory cells infiltration (Fig. 5c) and intramyocardial edema (Fig. 5d). Treatment diabetic rats with tempol normalized the alterations the cardiac tissues of these animals (Fig. 5e, f).

Fig. 5
figure 5

Representative micrographs of the left ventricle sections stained with hematoxylin and eosin illustrating effects of tempol on histopathological alterations induced by STZ in the heart tissues (magnification, ×400). n = 7 rats per group. a, b Micrographs of myocardium tissue sections of the control rats in the absence and presence of tempol, respectively are displaying normal histological structure. c, d Micrographs of cardiac tissue sections of diabetic rats exhibiting vaculations of cardiac myocytes associated with inflammatory cells infiltration along with intramyocardial edema. e, f Representative micrographs of the myocardium tissue sections revealing that tempol treatment attenuated the pathological alterations of the cardiac tissues of diabetic rats. Scale bar = 50 μm

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Table 2 Semiquantitative histological score of the cardiac sections
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As sensitive indicator of myocardial fibrosis, immunolocalization of TGF-β protein was detected by immunohistochemical staining with specific antibodies (Fig. 6). We here showed that TGF-β protein expression were markedly increased in the left ventricle of diabetic rats. Tempol significantly mitigated the observed upregulated TGF-β level in diabetic heart (Fig. 6).

Fig. 6
figure 6

Immunolocalization of TGF-β protein expression in left ventricles of STZ-induced diabetic rats. a Representative immunohistochemical micrograph analysis of TGF-β protein expression. b Bar graphs represent semiquantitative differences in expression of TGF-β expression. Values for each bar are the mean ± SEM; n = 7; *P < 0.01 vs. control group; #P < 0.01 vs. control + tempol-treated group; ƒP < 0.01 vs. STZ-treated group. Scale bar = 50 μm

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Discussion

Increased oxidative stress is believed to be an initial and important step in the development of cardiac dysfunction and cardiomyopathy (Norby et al. 2002; Ren and Davidoff 1997). Therefore, it is not surprising to attract considerable attention in our study. Data of the present study clearly demonstrate that tempol ameliorated diabetic cardiac fibrosis in rats via reducing the extracellular matrix, oxidative stress along with normalizing the MMP-2 activity thereby ameliorating the cardiac function.

ROS seems to be a direct consequence of hyperglycemia contributing to the development of fibrosis. ROS activates other signaling molecules such as mitogen-activated protein kinase, leading to the transcription of genes encoding cytokines, growth factors, and ECM proteins (Sano et al. 2001). Therefore, it convincible to hypothesize that ROS is involved in cardiac remodeling of diabetes indicating the benefits of ROS scavenging in the treatment of diabetic cardiomyopathy. Additionally, deficiency or inactivation of SOD that dismutates O2 − to H2O2 may determine an increased oxidative stress in cardiac tissues.

In this context, we observed that the SOD activity in the cardiac tissues of diabetic rats significantly decreased alongside increased ROS production as evidenced by lucigenin-enhanced chemiluminescence as compared with the control rats. This implied weak antioxidant status in diabetes, with difficulty to transform O2 − to H2O2.

Hereby, we postulated that tempol, as an efficient scavenger of free radicals would be predicted to be superior to other less-specific antioxidants for the treatment of cardiac fibrosis in diabetes. Herein, we explored the cardioprotective mechanisms of tempol by studying its effects on markers of oxidative stress. Interestingly, tempol exerted a marked reduction in ROS and this was associated with increase in the antioxidant enzyme capacity.

Conversely, fibrogenic cytokines have been recognized to regulate the extracellular matrix. TGF-β as an example of these cytokines acts as a profibrotic growth factor by upregulating the connective tissue growth factor through gene transcription (Massague and Chen 2000). In addition, TGF-β is increased in fibrotic diseases involved in cardiac fibrosis of diabetes (Martin et al. 2005; Tian et al. 2008). Furthermore, TGF-β can induce the production of collagen and fibronectin from cardiac fibroblasts and myocytes, and increased expression of this factor has been documented in the diabetic heart (Jesmin et al. 2002). Noteworthy, the current study revealed that oxidative stress in diabetic heart was associated with a marked increase in the cardiac TGF-β protein level.

As part of its profibrotic role, TGF-β enhances expression of ECM proteins such as fibronectin and collagen leading to cardiac fibrosis. Under diabetic conditions, excessive tissue fibrosis is regulated by high glucose levels, aldosterone, and TGF-β (Fang et al. 2004; Shimizu et al. 1993). Increased total collagen content in diabetic cardiomyopathy is accompanied with maladaptive changes in the composition of the extracellular cardiac matrix. Thus, it is tempting to speculate that the cardiac fibrosis is considered as a hallmark evidence of cardiomyopathy. Data of the present study confirmed this hypothesis and deduced that tempol exhibited anti-fibrotic action as indicated by its attenuation to the standard fibrotic markers, TGF-β and ECM.

Accumulation of cardiac fibrosis can result, on the one hand from excessive production of collagen by fibroblasts and on the other hand from decreased degradation of collagen by MMPs. MMPs are effectively involved in this turnover by degrading collagens in cardiac tissue (Visse and Nagase 2003). Numerous MMPs are known, but we focused on MMP-2 because of its effect on cardiac extracellular matrix. MMP-2 (72 kDa gelatinase and 72 kDa type IV collagenase) is able to degrade several ECM proteins including denaturated type IV collagen, the major structural component of basement membranes. Furthermore, a dysregulation of collagen degrading MMPs and their tissue inhibitors is supposed to be key regulator of myocardial fibrosis in diabetes (Wang et al. 2009).

Indeed, MMP-2 is involved in the pathogenesis of a wide variety of cardiovascular disorders (Ahmed et al. 2006; Pauschinger et al. 1999). Moreover, MMP-2 has recently been shown to be a direct mediator of ventricular remodeling and systolic dysfunction (Bergman et al. 2007). Several in vivo studies demonstrated that cardiac fibrosis in diabetic cardiomyopathy is associated with a decrease in MMP-2 expression/activity (Bollano et al. 2007; Van Linthout et al. 2008; Westermann et al. 2007). In consistent with these findings, a significant decrease in activity of MMP-2 in cardiac tissues of diabetic rats was observed. We, therefore, conclude that the decreased cardiac MMP-2 activity translates into less collagen degradation and thus promotes cardiac fibrosis. Tempol treatment was able to restore the MMP activity to the level of control. It is likely that oxidative stress in the heart of a diabetic rat may contribute to cardiac remodeling partly through decreasing cardiac gelatinolytic activity MMP-2. Hence, it is reasonable to suggest the ability of MMP-2 regulation to confer an additional protection thereby preventing cardiac remodeling. Additionally, tempol administration remarkably attenuated inflammatory responses evoked by STZ in cardiac diabetic tissues and abrogated the diabetic cardiac damage.

On the ground basis of the obtained data, we can suggest that there is a linked response between TGF-β/ECM axis and oxidative stress that ultimately contributed to development of the interstitial fibrosis in diabetes. Therefore, we should see them as a potential therapeutic target, and it is meaningful to prevent myocardial damage.

Conversely, the CK-MB is part of total CK and more specific for cardiac muscle than other striated muscle. Serum CK-MB and LDH levels were also reported to increase in STZ-induced diabetic rats, and may serve as a marker for cardiovascular risk and cardiac muscular damage (Gohlke et al. 1994). In this setting, we observed significant increases in LDH and CK levels in STZ-induced diabetic rats as compared with normal rats. Tempol treatment markedly reduced LDH and CK levels and further substantiates its beneficial effect by reducing the diabetic cardiomyopathy.

Cardiac fibrosis is one of the main modulators of diastolic cardiac stiffness (Zile and Brutsaert 2002). It has been shown that hypertension was associated with development of bradycardia in diabetic rat model (Rodrigues et al. 1986). Fibrosis decreases myocardial compliance and causes cardiac dysfunction. In the current study, we observed that our experimental model showed a decrease in HR, which was associated with increased SBP. However, the reason for this observation so far remains unclear. Although it is well known that hypertension is one of the major cardiovascular complications in diabetic patients, there are contradictory results as to whether STZ-induced diabetic rats become hypertensive because of methods used to measure blood pressure. STZ-diabetic rats were found to be hypertensive (El-Bassossy et al. 2011; Goyal et al. 1998; Sowers and Epstein 1995) and normotensive (Yamamoto 1988) or hypotensive (Kusaka et al. 1987) when BP was measured indirectly by using tail cuff method and directly via an arterial cannula. These discrepancies in the literature regarding BP changes in STZ-induced diabetic rats may be due to differences in age and time of experimentation as well as the methodology for BP measurement. Importantly, treatment with tempol significantly reduced blood pressure and corrected the heart rate in addition to improvement of cardiomyopathy markers such as CK-MB and LDH as compared with the diabetic control group.

It is widely accepted that oxidants can stimulate both the accumulation of collagen and extracellular matrix deposition by modulating the expression of inflammatory cytokine genes, alongside inducing the expression and synthesis of fibrogenic cytokines. Thus, it is reasonable to suggest that the observed lower SBP in tempol-treated diabetic rats may be mediated, at least in part, by the maintenance of antioxidant status within the physiological range. However, it remains unclear whether tempol only can directly act on the myocardium or the reduced STZ-induced remodeling due to lowering of SPB by tempol treatment may contribute to its beneficial effects.

Nevertheless, the present study has some limitations: (1) despite the importance of SBP and heart rate in the general context of cardiovascular dysfunction and its pathological consequences, lack of the direct cardiac function parameters is among limitations of the present study. (2) Although we have estimated collagen deposition by Western blot analysis, there was a lack in quantification of left ventricular fibrosis by Sirius red staining.

Taken together, data of the present study demonstrate that tempol has a potential benefit in alleviation of diabetes-associated cardiac fibrosis and adds a novel cardioprotective effect in STZ-induced diabetic rats. Parallel reductions in ECM, TGF-β, and ROS levels and restoring the antioxidant enzyme activity alongside normalization of MMP-2 activity have been suggested to be among the major factors of this protection. A better understanding of these mechanisms could be of importance as a useful therapeutic target in the management of diabetic cardiac fibrosis.